Cytotoxic T lymphocytes (xe2x80x9cCTLxe2x80x9d) represent an important component of an animal""s immune response against a variety of pathogens and cancers. CTL which have been specifically activated against a particular antigen are capable of killing the cell that contains or expresses the antigen. CTL are particularly important in providing an effective immune response against intracellular pathogens, such as a wide variety of viruses, and some bacteria and parasites. CTL responses are also believed to be capable of contributing to anti-tumor responses in afflicted or susceptible individuals.
The receptors on the surface of the CTL cannot recognize a foreign antigen directly, however. The CTL express an xcex1-xcex2 heterodimeric T cell receptor which is capable of recognizing foreign antigen fragments bound to major histocompatibility complex (MHC) class I molecules on the surface of the effected (e.g., infected) cells. CTL also express the non-polymorphic CD8 antigen. This cell surface protein interacts with the third domain of the class I molecule on the antigen presenting cells and plays a role in both stabilizing the interaction between the CTL and the antigen presenting cell and in CTL activation (Salter et al., Nature 345:41-46 (1990)).
There are a number of mechanisms by which CTL are thought to disrupt the infectious or tumorigenic process. Among these, one involves the production of lymphokines such as gamma interferon (IFNxcex3) and tumor necrosis factor alpha (TNFa), which are known to act directly on infected cells to inhibit viral replication (Gilles et al., J. Virol. 66:3955-3960 (1992)). In addition, IFNxcex3 causes increased expression of MHC class I molecules on the surface of virus infected cells and enhances their ability to be recognized by CTL and trigger immune intervention (Hayata et al., Hepatology 13:1022-1028 (1991)).
A second mechanism by which CTL combat infections or tumors is through direct killing of the afflicted cell, e.g., those which are infected by the targeted virus (Cohen et al., Ann. Rev. Immunol. 10:267-293 (1992) and Henkart et al., Ann. Rev. Immunol. 3:31-58 (1985)). For example, since viruses must replicate within the host cell the lysis of infected cells destroys virus production prior to the liberation of infectious particles. The exact mechanism(s) by which CTL kill infected target cells remains unclear. Once CTL recognized an antigen presenting cell, close contact between the cells is established over a large surface area. A xe2x80x9cdirect hitxe2x80x9d is then delivered by translocating enzymes present in cytoplasmic vacuoles of CTL to the antigen presenting cell, which enzymes kill the cell or perhaps induce programmed cell death, xe2x80x9capoptosisxe2x80x9d. Once CTL have delivered their xe2x80x9clethal hitxe2x80x9d to the antigen presenting cells, they can detach and go on to kill other antigen presenting cells through repetition of the antigen-specific recognition, lymphokine release and target cell killing mechanisms.
The means by which CTL distinguish infected from non-infected cells is through the T cell receptor and its ability to specifically recognize a peptide fragment of viral protein that is bound to the peptide-binding cleft of the MHC class I molecule (Monaco et al., Immunol. Today 13:173-179 (1992) and Townsend et al., Ann. Rev. Immunol. 7:601-624 (1989)). Several viral fragments that can serve an antigenic peptides have been identified.
The biochemical events that take place in the cytoplasm of infected cells leading to CTL recognition are termed antigen processing and presentation. While not completely defined, it seems clear that during the synthesis and assembly of the infecting viral or bacterial proteins, some proteolysis takes place in the cytoplasm (Monaco et al., Immunol. Today 13:173-179 (1992)). Structures called proteosomes cleave the foreign proteins into peptide fragments. These fragments are then transported into the endoplasmic reticulum (ER) by means of specific transporter proteins where newly synthesized MHC class I molecules are present. Those peptides that are capable of specifically binding to a given MHC class I molecule do so in the ER. The non-polymorphic class I xcex2 chain, xcex22 microglobulin binds to the antigenic peptide-class I complex, thus forming a stable trimolecular complex that is transported to the cell surface and displayed as an integral membrane component.
The selection of which peptides bind to a particular MHC class I molecule is based on the ability of the peptide to bind within the binding pocket or cleft which resides at the outermost apex of the extra-cellular portion of the MHC molecule. For several MHC molecules, this peptide binding pocket has been precisely defined by X-ray crystallographic procedures allowing a visualization of the types and location of the chemical bonds that form to stabilize the interaction (Saper et al., J. Mol. Biol. 219:277-319 (1991)).
Because of the differences in the structure of the peptide binding pocket between the diverse set of histocompatibility alleles, e.g., the human HLA alleles, a distinct population of antigenic peptides is bound by each allele, although in some cases the population of antigenic peptides may overlap for closely related alleles. Thus, the specificity of the CTL for a foreign antigen resides at the level of the ability of MHC class I molecules to bind to a specific peptide as well as for the T cell receptor on the CTL to recognize the foreign protein fragment bound to that specific MHC class I allele.
In animals, CT8+, MHC class I-restricted cytotoxic T cells play an important role in the immune mediated clearance of viral infections (e.g., Oldstone et al., Nature 321:239-243 (1986); Mackenzie et al., Immunol. 67:375 (1989); and Robertson et al., J. Virol. 66:3271-3277 (1992)). While similar studies have not been possible in humans, and thus direct proof is still lacking, all of the evidence points to a similar role for CTL.
The importance of CTL in viral clearance in animals is evidenced by lymphocytic choriomeningitis virus (LCMV) infection in mice (Oldstone et al., Nature 321:239-243 (1986); Mackenzie et al., Immunol. 67:375 (1989); Robertson et al., J. Virol. 66:3271-3277 (1992); and Ahmed et al., J. Virol. 61:3920-3929 (1987)). When LCMV infects newborns or immune-suppressed adult animals, they become chronically infected and virus is expressed in nearly all tissues of the body. In contrast, adult mice infected with LCMV mount a vigorous cellular and humoral response against the virus and clear the infection within one to two weeks. When chronic carriers of LCMV are adoptively treated by transfer of CD8+LCMV-specific, MHC class I-restricted CTL, the viral infection is cleared and the mice become resistant to subsequent LCMV challenge. Additional studies have shown that CTL are necessary and sufficient for LCMV clearance and that other aspects of the immune system need not be functioning (Oldstone et al., Nature 321:239-243 (1986) and Schulz et al. Proc. Natl. Acad. Sci. USA 88:991-993 (1991)).
In addition to mediating the clearance of virus from chronically infected animals, studies have demonstrated that CTL generated in vivo against a synthetic peptide which presents an antigenic epitope of LCMV are able to protect mice against acute infection (Schulz et al., Proc. Natl. Acad. Sci. USA 88:991-993 (1991)). Mice injected with 100 xcexcg of a synthetic 15 amino acid peptide in complete Freund""s adjuvant were fully protected from a lethal LCMV challenge.
With regard to the role of CTL in other viral infections, studies with influenza virus and respiratory syncytial virus in mice have similarly demonstrated the importance of CTL activation in the rapid and effective recovery from these infections.
Strong evidence from animal studies indicates that an acute infection can become chronic when there is an inadequate immune response to clear the infection (Ahmed, Concepts in Viral Pathogenesis III, Notkins and Oldstone eds., Springer-Verlag, New York, 304-310 (1989)). Once the chronic infection has been established, it appears to be more or less xe2x80x9ctoleratedxe2x80x9d by the host""s immune system. Tolerance appears to be organism-specific rather than a result of general immunosuppression (Fields et al., Fields Virology, Raven Press, New York, N.Y. 2:2137-2236 (1990)). Studies examining which cells in the immune system are anergic or tolerant to the infecting organism suggest that the CD4+, class II-restricted T xe2x80x9chelperxe2x80x9d cells are dysfunctional (Schwartz, Cell 57:1073-1081 (1989)). Since class II-restricted T helper cells play a critical role in the initial priming of class I-restricted CTL (Cassell et al., Ann. NY Acad. Sci. 532:51-60 (1988) and Fayolle et al., J. Immunol. 174:4069 (1991)), diminished CD4 cell function may impair the capacity of the immune system to respond adequately, and may thus clear the way for chronic infection.
Decreased T helper cell activity has been shown in the case of chronic hepatitis B infection in humans, although the fact that some CD4+ T helper function is seen suggests that these cells are not completely dysfunctional (see, e.g., Ahmed et al., J. Virol. 61:3920-3929 (1987); Alberti et al., Lancet 1:1421-1424 (1988); Neurath et al., Nature 315:154-156 (1985); Celis et al., J. Immunol. 132:1511-1516 (1984); and Ferrari et al., J. Immunol. 139:2050-2055 (1987)). Class I-restricted CTL can be detected in patients with chronic HBV infection (Barnaba et al., J. Immunol. 143:2650-2654 (1989)).
The requirement for lymphokines such as IL-2 in the generation of CD8+ CTL is well established, although the need for activation of CD4+ T helper cells to provide these lymphokines remains somewhat controversial. While the concept of linked T helper-B cell recognition for antibody production has been firmly defined, there is no compelling evidence for linked T helper-CTL recognition for the in vivo induction of CD8+ CTL. See, e.g., Buller et al., Nature 328:77-79 (1987); Sarobe et al., Eur. J. Immunol. 21:1555-1558 (1991); and Cassell and Forman, Annals N.Y. Acad. Sci. 51-60 (1991).
Thus, the data available suggest that CD8+ class I-restricted cytotoxic T cells specific for foreign antigens such as viral proteins play a critical part in prevention of disease and clearance of an established disease process. Therefore, the challenge is to induce a sufficiently potent, antigen-specific, cell-mediated immune response in humans and other mammals which, by itself or in conjunction with chemotherapeutic agents or the like, will either prevent a disease process such as an infection or tumor from becoming established, or will eliminate or at least ameliorate an infection or tumor which has already become established in the host. Quite surprisingly, the present invention fulfills these and other related needs.
The present invention provides compositions for inducing a cytotoxic T lymphocyte response to an antigen of interest in a mammal. The compositions comprise a peptide that induces a CTL response to the antigen and a peptide that induces a HTL response, wherein the HTL-inducing peptide is lipidated. The HTL-inducing peptide is optionally linked to the CTL-inducing peptide or not linked. When linked, the HTL-inducing peptide may be separated from the CTL peptide by a spacer, such as Ala-Ala-Ala. The HTL-inducing peptide will usually be linked at its C-terminal end to the CTL-inducing peptide. Typically, the lipid is linked to the N-terminus of the HTL-inducing peptide, where the linkage can optionally include a spacer, such as Lys-Ser-Ser or the like.
The antigen to which the cytotoxic T lymphocyte response is induced is selected from a viral, bacterial, parasitic or tumor antigen. Among the viral antigens to which the CTL responses are effectively induced are antigens of hepatitis B (such as envelope, core or polymerase antigens), hepatitis C or human papilloma virus. A particularly effective hepatitis B antigen is HBc18-27. Typically the CTL inducing peptide will be from seven to fifteen residues, and more usually from nine to eleven residues. The immunogenic composition can further comprise a carrier, such as physiologic saline, and an adjuvant, such as incomplete freunds adjuvant, alum or montanide. When the peptide is lipidated, it may be modified or unmodified. The lipid is preferably a linear alkyl chain of 6-22 carbons, and preferably is a linear alkyl chain of 16 carbons. In some embodiments of the present invention the lipid is comprised of palmitic acid attached to epsilon and alpha amino groups of a Lys residue, wherein the Lys is linked to the amino terminus of the HTL-inducing peptide by means of a linker.
In other embodiments the present invention comprises methods for inducing a cytotoxic T lymphocyte response in a mammal against an antigen such as a viral, bacterial, parasitic, or tumor antigen. The method comprises administering to the mammal a peptide that induces a CTL response to the antigen, and administering, either separately or together, a lipidated peptide that induces a HTL response. The HTL-inducing peptide is optionally linked to the CTL-inducing peptide or unlinked. When unlinked, the HTL-inducing peptide can be admixed with the CTL-inducing peptide. The HTL-inducing peptide and the CTL-inducing peptide are typically administered to the mammal in a regimen of two or more administrations. These boosters are spaced a sufficient interval apart to optimize development of a CTL response to the antigen of interest, e.g., a second administration may be approximately four weeks after the initial administration. In representative embodiments described herein the antigen is hepatitis B antigen, such as HBc18-27 and the mammal is a human, of the HLA-A2.1 histocompatibility type for the HBc18-27 CTL inducing peptide.
In yet other embodiments the invention provides methods for treating or preventing a disease that is susceptible to treatment by a CTL response by administering a CTL-inducing peptide to an antigen associated with said disease, and a HTL-inducing peptide conjugated to a lipid. The induction of a CTL response can be used in the treatment or prevention of viral infection (e.g., hepatitis B, hepatitis C or human papilloma virus), bacterial or parasitic infection or tumors. When the disease is hepatitis B infection, for example, the methods can be used to treat or prevent chronic or acute infection.
In yet other embodiments the invention provides methods for inducing a cytotoxic T lymphocyte response in a human against an antigen of interest. The methods comprise administering a composition which comprises a peptide that induces a CTL response to said antigen in a human and an adjuvant. The method may further comprise administering a peptide that induces a HTL response, and in some embodiments the HTL inducing peptide is linked to the CTL inducing peptide.
In other aspects the invention provides methods for inducing a CTL response in a human against an antigen of interest by administering a peptide that induces a CTL response to the antigen and a peptide that induces a HTL response, where the CTL inducing and/or the HTL inducing peptide is lipidated. The CTL and HTL inducing peptides may be linked or unlinked. The HTL inducing peptide is preferably lipidated. The lipidated HTL inducing peptide can be combined with a cocktail of at least two CTL inducing peptides to optimize coverage of individuals of different HLA types or, in some instances, different antigen strains.
In a further aspect of the invention methods are described for inducing an effective CTL response in a human against an antigen of interest. According to these methods one or more peptides that induce a CTL response to the antigen, such as a viral, bacterial, parasitic or tumor antigen, is administered to a human together or separately with a peptide that induces a HTL response, where at least the CTL inducing and/or the HTL inducing peptide is lipidated. In representative embodiments of such a method described herein the CTL response is induced to a viral antigen, such as hepatitis B antigen.
Pharmaceutical composition for the treatment of hepatitis B infection are also provided. These compositions comprise a peptide that induces a CTL response to hepatitis B and a peptide that induces a HTL response, where the HTL-inducing peptide is conjugated to a lipid, together with a pharmaceutically acceptable carrier. The carrier can be a liposome, for example, and the pharmaceutical composition may further comprises an adjuvant, such as incomplete Freund""s adjuvant, alum or MONTANIDE(copyright) (Seppic, Paris, France; oil-based adjuvant with mannide oleate).